CN114853828A

CN114853828A - Compounds, conjugates and uses thereof

Info

Publication number: CN114853828A
Application number: CN202110073911.7A
Authority: CN
Inventors: 张必良; 赵浩廷
Original assignee: Agna Biopharmaceutical Co ltd
Current assignee: Agna Biopharmaceutical Co ltd
Priority date: 2021-01-20
Filing date: 2021-01-20
Publication date: 2022-08-05

Abstract

The invention provides a compound, which has a structure shown in a formula (I) and a formula (II). The invention also provides conjugates of the compounds with drug molecules, and uses of the compounds and conjugates, such as in detection and therapy.

Description

Compounds, conjugates and uses thereof

Technical Field

The present invention relates to the field of biotechnology, in particular, the present invention relates to compounds, conjugates and uses thereof, and more particularly, the present invention relates to compounds, conjugates, methods of preparing compounds, compositions and uses thereof.

Background

Asialoglycoprotein receptor (ASGPR) is an abundant endocytotic receptor of a hetero-oligomer, mainly present on the cell membrane surface of the liver parenchymal cell toward the side of the sinusoid, having specific recognition of sugars, and since various glycoproteins after enzymatic or acid hydrolysis to remove terminal sialic acid expose the exposed minor terminal galactose residue, the sugar binding specificity of ASGPR actually resides in galactosyl groups and is also called galactose-specific receptor. ASGPR is mainly distributed in liver parenchymal cells, and other cells are low in content, so that ASGPR becomes an optimal receptor for liver directional transport.

Glycoproteins terminating in a non-reducing galactose (Gal) or N-acetylgalactosamine (GalNAc) residue are both recognized by ASGPR, GalNAc binding ASGPR with approximately 50-fold greater affinity than Gal (lobst STet al, J Biol Chem, 1996, 271(12): 6686-6693). In vitro experiments have shown that clustered sugar residues can have much higher affinity than non-clustered sugar residues by simultaneously occupying the binding site of the receptor, in the order of affinity: tetraantenna > triantenna > > biantenna > monocrotarside galactosides (Lee YC, et al, J Biol Chem, 1983, 258(1): 199-.

The ASGPR receptor mediated liver targeting oligonucleotide is a new breakthrough in the research field of nucleic acid innovative drugs. In 2012, the american pharmaceutical company covalently linked the triantenna GalNAc structure of the previous study to small interfering rna (siRNA), achieving liver-targeted delivery of siRNA in vivo. By applying the technology, researchers develop the drug development of diseases such as amyloidosis, hemophilia, hypercholesterolemia, hepatic porphyrin, hepatitis B and the like, the first GalNAc-siRNA drug is on the market in 2019, two drugs are applying for the market, and more than ten candidate drugs enter clinical research (http:// www.alnylam.com/product-pipeline /). In 2014, the American ISIS pharmacy covalently links the triantenna GalNAc and antisense Nucleic acid to realize liver targeting administration in animals, and the activity of the antisense Nucleic acid is improved by 10 times after the linkage (Prakash TPet al, Nucleic Acids Res.42, 8796-807).

However, the ASGPR receptor-mediated liver targeting oligonucleotide technology still needs further development and improvement.

Disclosure of Invention

The applicant of the present application uses Pentaerythritol (PET) as a main structure to form a multi-antennal ASGPR ligand, wherein one of the four hydroxyl groups of Pentaerythritol can be converted into various functional groups, such as amino, azide, carboxyl, carbonyl and the like, and can be linked with various chemical groups to form a diversified linked GalNAc receptor. Compared with the multi-antenna ASGPR ligand formed by taking TRIS (TRIS) as a main structure, which is a product of Alylam company, the multi-antenna ASGPR ligand can obtain GalNAc coupled oligonucleotides with more diversified structures.

Based on this, in a first aspect of the invention, the invention proposes a compound. According to an embodiment of the invention, the compound has the structure shown in formula (I),

wherein R is ₁ Is a moiety that binds to ASGPR; r ₂ is-CH ₂ -、

C _6-14 Aryl radical, C _2-6 Alkenyl or C _2-6 An alkynyl group; r is ₃ Is composed of

-O-、C _6-14 An aromatic group or a 3-to 10-membered heterocyclic group; r ₄ is-CH ₂ -、

C _6-14 Aryl or 3 to 10 membered heterocyclyl; r ₅ is-OH,

-COOH、

PG is a carboxyl protecting group; PG (Picture experts group) ₂ Is a hydroxy protecting group; a. b, c and d are each independently any integer from 1 to 10; n is any integer of 1-3. The compound shown in the formula (I) according to the embodiment of the invention can be connected with a plurality of oligonucleotide chains through phosphodiester bonds, the connecting position can be between the 5 'end, the 3' end or the 5 'end and the 3' end of the oligonucleotide chains, the types of the connectable oligonucleotides are greatly expanded compared with the prior art, the non-limited connecting position provides more choices for the modification of the oligonucleotides, and the improvement of the pharmacokinetic properties of nucleic acid drugs is facilitated.

According to an embodiment of the present invention, the above compound may further comprise at least one of the following additional technical features:

according to an embodiment of the present invention, R ₁ The end of the part is galactose or N-acetylgalactosamine;

according to an embodiment of the invention, said R ₁ Moieties are selected from the following structures:

wherein q and q' are each independently any integer from 1 to 10.

According to an embodiment of the invention, the carboxyl protecting group is selected from the group consisting of succinate, pentafluorophenol ester, and,

Wherein h is any integer from 1 to 5.

According to an embodiment of the invention, the hydroxyl protecting group is selected from silyl, monomethoxytrityl (MMTr), 4' -dimethoxytrityl (DMTr) and trityl.

According to an embodiment of the invention, the silyl group is selected from the group consisting of tert-butyldimethylsilyl ether (TBMDS), tert-butyldiphenylsilyl (TBDPS) and triisopropylsilyl ether (TIPS).

According to an embodiment of the invention, the compound has the structure shown below:

in a second aspect of the invention, the invention features a compound. According to an embodiment of the present invention, the compound has a structure represented by formula (II),

wherein R is ₁ Is a moiety that binds to asialoglycoprotein receptor (ASGPR); r ₂ is-CH ₂ -、

C _6-14 Aryl radical, C _2-6 Alkenyl or C _2-6 An alkynyl group; a and b are each independently any integer from 1 to 10; n is any integer of 1-3.

The compound shown in the formula (II) according to the embodiment of the invention can be used as a coupling reagent to be directly coupled with an alkynyl-containing oligonucleotide, and also can be used as a precursor of the coupling reagent to be subjected to mild and efficient reaction with a compound with an active group, such as an alkynyl-containing pentafluorophenol ester and the like, so as to obtain a Gal or GalNAc coupling reagent of the pentafluorophenol ester, and further coupled with oligonucleotides with various functional groups, so as to obtain Gal or GalNAc coupled oligonucleotides with more diversified structures.

according to an embodiment of the invention, R ₁ The end of the part is galactose or N-acetylgalactosamine.

wherein q and q' are each independently any integer from 1 to 10.

in a third aspect of the invention, the invention features a conjugate. According to an embodiment of the invention, the conjugate has a structure represented by formula (III),

wherein R is ₁ Is a moiety that binds to ASGPR; r ₂ is-CH ₂ -、

C _6-14 Aryl or 3 to 10 membered heterocyclyl; r is O or S; rx is a drug molecule selected from the group consisting of cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, toxins, radioisotopes, and oligonucleotide chains; a. b, c and d are each independently any integer from 1 to 10; n is any integer of 1-3.

According to an embodiment of the invention, the above-mentioned conjugate may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the conjugate has a structure represented by formula (IV),

formula (IV), wherein the oligo is a strand of oligonucleotides, optionally linked as a single-stranded oligonucleotide or a double-stranded oligonucleotide or a combination thereof.

In a fourth aspect of the invention, the invention features a conjugate. According to an embodiment of the invention, the conjugate is formed by linking a compound of the first or second aspect of the invention to a drug molecule selected from the group consisting of cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, toxins, radioisotopes, and oligonucleotide chains.

The conjugates according to embodiments of the third or fourth aspect of the invention are capable of specifically targeting the liver, and their conjugated drug molecules, such as oligonucleotide strands, are specifically introduced into hepatocytes to effect intervention or detection of a particular gene in the hepatocytes, or to effect treatment or prevention of a pathological condition or disease caused by expression of a particular gene in the hepatocytes.

According to an embodiment of the invention, the drug molecule is an oligonucleotide strand, optionally the oligonucleotide strand is a single stranded oligonucleotide, a double stranded oligonucleotide or a combination thereof.

According to an embodiment of the conjugate of the third or fourth aspect of the invention, the conjugate may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the oligonucleotide strand comprises unmodified nucleotides and/or modified nucleotides.

According to an embodiment of the invention, the modified nucleotides are each independently selected from the group consisting of 2' -methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-allyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxy modified nucleotides, 2' -hydroxy modified nucleotides, locked nucleotide modified nucleotides, Glycerol Nucleic Acid (GNA) modified nucleotides and unlocked nucleic acid modified nucleotides (UNA).

According to an embodiment of the invention, the 2 '-O-alkyl modification is a 2' -O-methyl modification.

According to an embodiment of the invention, the oligonucleotide strand has a terminal modification.

According to an embodiment of the invention, the end-modifier is selected from cholesterol, polyethylene glycol, a fluorescent probe, biotin, a polypeptide, a vitamin, a tissue targeting molecule, or a combination thereof.

According to an embodiment of the invention, the length of the oligonucleotide strand is between 5 and 100 bp.

According to an embodiment of the invention, the compound is linked to the 5 'terminus, the 3' terminus or any nucleotide between the 5 'terminus and the 3' terminus of at least one strand of the oligonucleotide strand by a phosphoester linkage. Compared with the prior art, the types of the connectable oligonucleotides are greatly expanded, and the non-limited connection positions provide more choices for the modification of the oligonucleotides, thereby being beneficial to improving the pharmaceutical properties of nucleic acid drugs.

According to an embodiment of the invention, the phosphate ester bond is a phosphodiester bond or a modified phosphate ester bond.

According to an embodiment of the invention, the modified phosphate ester bond is selected from the group consisting of a thio-modified phosphate ester bond, an amino-modified phosphate ester bond.

According to an embodiment of the invention, the ribose-phosphate backbone of the oligonucleotide strand is substituted with a Polypeptide Nucleic Acid (PNA), or a morpholine ring antisense nucleotide (PMO).

According to an embodiment of the invention, the conjugate is obtained by solid phase synthesis or liquid phase synthesis.

In a fifth aspect of the invention, a process for preparing a compound of formula (II) is provided. According to an embodiment of the invention, the method comprises reacting pentaerythritol, NaN ₃ And carrying out a synthesis reaction of galactose or a galactose derivative to obtain the compound.

The method according to the embodiment of the invention adopts Pentaerythritol (PET) as a main structure for preparing the multi-antenna ASGPR ligand for the first time, one of four hydroxyl groups is converted into azide, the prepared product can be directly coupled with the oligonucleotide containing alkynyl, and can also be subjected to mild and high-efficiency reaction with compounds with active groups such as pentafluorophenyl ester containing alkynyl to obtain a Gal or GalNAc coupling reagent of the pentafluorophenyl ester, and further coupled with oligonucleotides with various functional groups to obtain Gal or GalNAc coupled oligonucleotides with diversified structures.

In a sixth aspect of the invention, a process for preparing a compound of formula (I) is provided. According to an embodiment of the invention, the method comprises contacting a first compound with a second compound so as to obtain the compound, wherein the first compound is as defined in the second aspect or obtained according to the method of the fifth aspect.

According to a particular embodiment of the invention, the second compound has the structure shown below:

wherein c and d are each independently any integer from 1 to 10.

According to the method disclosed by the embodiment of the invention, the compound shown in the formula (II) and the alkynyl-containing compound with the active group are subjected to mild and efficient reaction, and then a Gal or GalNAc coupling reagent with the active group can be obtained; or a compound of formula (II) in H ₂ In the presence of a reactive group, the azido group is converted to an amino group, which in turn reacts with a second compound to form a Gal or GalNAc coupling reagent with a reactive group. The compound prepared by the method according to the embodiment of the invention can be connected with a plurality of oligonucleotide chains through phosphodiester bonds, the connecting position can be between the 5 'end, the 3' end or the 5 'end and the 3' end of the oligonucleotide chains, the types of the connectable oligonucleotides are greatly expanded compared with the prior art, the non-limited connecting position provides more choices for the modification of the oligonucleotide chains, and the improvement of the pharmaceutical properties of nucleic acid drugs is facilitated.

In a seventh aspect of the invention, a composition is provided. According to an embodiment of the invention, the composition comprises: a conjugate according to the third or fourth aspect of the invention. The composition according to the embodiment of the invention can specifically target the liver cells, so that the coupled drug molecules, such as oligonucleotides, enter the liver cells, and the intervention or detection of genes in the liver cells is realized.

According to an embodiment of the present invention, the above composition may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the composition further comprises a pharmaceutically acceptable carrier or excipient.

According to an embodiment of the present invention, the composition is in the form of powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol, powder spray or microneedle patch.

According to an embodiment of the invention, the composition is administered to the subject by intravenous, intramuscular, subcutaneous injection.

According to an embodiment of the invention, the subject is a mammal.

According to a particular embodiment of the invention, the mammal is selected from the group consisting of bovine, equine, ovine, porcine, canine, feline, rodent and primate.

In an eighth aspect of the invention, the invention provides the use of a conjugate of the third or fourth aspect in the manufacture of a medicament for the treatment and/or prevention of a pathological condition or disease associated with expression or overexpression of a gene in a hepatocyte. The composition according to the embodiment of the invention can specifically target the liver cells, so that the coupled drug molecules, such as oligonucleotides, enter the liver cells, and the intervention of genes in the liver cells is realized. The medicament prepared by the composition according to the embodiment of the present invention can be used for effective treatment and/or prevention of pathological conditions or diseases associated with expression or overexpression of genes in hepatocytes.

According to a particular embodiment of the invention, the gene is selected from the group consisting of HBV genome, HCV genome, PCSK9, xanthine oxidase, URAT1, APOB, liver fibrosis related genes (AP3S2, AQP2, AZINl, DEGSl, STXBP5L, TLR4, TRPM5, etc.), non alcoholic steatohepatitis (PNPLA3, FDFTl), primary biliary cirrhosis (HLA-DQB1, IL-12RB2, etc.), or a combination thereof.

According to a particular embodiment of the invention, the disease is selected from hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, defects in bile acid synthesis and metabolism, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, Hepatitis B (HBV), Hepatitis C (HCV), steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia or a disease involving abnormally increased hepatic glucogenesis similar to type II diabetes, hepatitis and hepatic porphyrins.

In a ninth aspect, the invention provides the use of a compound as described in the first or second aspects above or a conjugate as described in the third or fourth aspects, in the preparation of a composition for liver targeting.

In a tenth aspect of the invention, the invention provides the use of a conjugate of the third or fourth aspect in the preparation of a composition for liver-targeted RNA detection or localization.

In an eleventh aspect of the invention, the invention provides a conjugate of the third or fourth aspect for use in the treatment and/or prevention of a pathological condition or disease associated with expression or overexpression of a gene in a hepatocyte.

According to an embodiment of the invention, the gene is selected from the group consisting of HBV genome, HCV genome, PCSK9, xanthine oxidase, URAT1, APOB, liver fibrosis related genes (AP3S2, AQP2, AZINl, DEGSl, STXBP5L, TLR4, TRPM5, etc.), non alcoholic steatohepatitis (PNPLA3, FDFTl), primary biliary cirrhosis (HLA-DQB1, IL-12RB2, etc.), or a combination thereof.

According to an embodiment of the invention, the disease is selected from hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, defects in bile acid synthesis and metabolism, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, Hepatitis B (HBV), Hepatitis C (HCV), steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia or a disease involving abnormally increased hepatic glucogenesis similar to type II diabetes, hepatitis and hepatic porphyrins.

In a twelfth aspect of the invention, the invention provides a compound of the first, second or third or fourth aspect for use in liver targeting.

In a thirteenth aspect of the invention, the invention provides a conjugate of the third or fourth aspect for liver-targeted RNA detection or localization.

In a fourteenth aspect of the invention, the invention proposes a method of treating and/or preventing a pathological condition or disease associated with the expression or overexpression of genes in hepatocytes. According to an embodiment of the invention, the method comprises administering to the subject a pharmaceutically acceptable amount of the conjugate of the third or fourth aspect.

In a fifteenth aspect of the invention, a method of intervening in or detecting a predetermined gene in a hepatocyte is presented. According to an embodiment of the invention, the method comprises: contacting a cell population comprising said hepatocytes with the conjugate of the third or fourth aspect, wherein the oligonucleotide strands in the conjugate interact with said predetermined gene. According to the method provided by the embodiment of the invention, the conjugate is specifically targeted to the liver cell, and the oligonucleotide chain coupled with the conjugate enters the liver cell, so that the intervention or detection of genes in the liver cell is realized. It should be noted that the manner of "the oligonucleotide chain in the conjugate interacts with the predetermined gene" described herein is not particularly limited, and the interaction manner may be direct or indirect, and includes, but is not limited to, binding, complementary pairing, and the effect generated after the interaction is adapted to the interaction mode of the two, for example, when the conjugated oligonucleotide chain is siRNA against the predetermined gene, the silencing of the predetermined gene can be realized after the interaction, for example, when the conjugated oligonucleotide chain is probe against the predetermined gene, the localization or detection of the predetermined gene can be realized after the interaction.

Compared with the prior art, the invention has the following remarkable differences and technical progress:

1. the chemical structure is different. In the prior art, trihydroxymethyl aminomethane (TRIS) is used as a main structure to form a multi-antennary ASGPR ligand, Pentaerythritol (PET) is used as a main structure to form the multi-antennary ASGPR ligand, one of four hydroxyl groups can be converted into various functional groups, such as amino, azide, carboxyl, carbonyl and the like, and can be subjected to a connection reaction with various chemical groups to form a diversified GalNAc receptor. For example, azido-PETGalNAc can be coupled with an alkynyl-containing oligonucleotide chain, can be mildly and efficiently reacted with a compound with an active group such as an alkynyl-containing pentafluorophenol ester to obtain a GalNAc coupling reagent of the pentafluorophenol ester, and can be directly coupled with oligonucleotides with various functional groups to obtain GalNAc coupled oligonucleotides with diversified structures.

2. The synthesis method is more concise and efficient. The target molecule can be obtained by one-step click reaction of the obtained azide and branched chain alkynyl, reaction steps are reduced, synthesis efficiency is improved, and a reaction route, an experimental process and subsequent purification are simpler and easy to operate;

3. the covalent attachment of ligands to oligonucleotide strands is different. In the prior art, a three-antennary GalNAc ligand is connected with a solid phase support, and the modified solid phase support is used for oligonucleotide solid phase synthesis, so that the ligand can only be connected at the 3' terminal of oligonucleotide. The invention can connect phosphoramidite of the triantenna GalNAc ligand to the 5 ' terminal oligonucleotide by solid phase synthesis, and can also complete solid phase synthesis, (1) the active ester such as pentafluorophenol ester of the triantenna GalNAc ligand and the like are connected with amino modified oligonucleotide by liquid phase connection, and the triantenna PETGAlNAc ligand can be connected to the terminal (3 ' or 5 ') or any nucleotide middle position of the oligonucleotide; (2) by azido (N) groups of the triantenna GalNAc ligands ₃ -PETGAlNAc) and the terminal or any intermediate position of the ethynyl modified oligonucleotide liquid phase connection method, the method has mild reaction, high coupling yield and simple operation; (3) multiple ligand modifications can be achieved by coupling to multiple amino or ethynyl oligonucleotides. The invention enlarges the modification variety of the oligonucleotide, is beneficial to finding the patent medicine molecules with higher activity, and is more suitable for large-scale production.

4. The oligonucleotide sites can be modified differently and in different combinations. Terminal sites in oligonucleotide drug molecules are usually modified with cholesterol, polyethylene glycol (PEG), and the like, to improve pharmacokinetic properties. The triantenna GalNAc ligand designed by the prior art is only used for modifying the 3' -end of an oligonucleic acid chain, occupies an end modification site, and reduces the types of oligonucleic acid modification. The novel compounds of the invention can be modified at any position on the oligonucleotide, other modifications being unaffected by the terminus. The preparation of new compounds that modify oligonucleotide chains in combination with terminal cholesterol is described in embodiments of the invention.

Detailed Description

The following examples are provided to further illustrate the invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The terms used herein

N in the formulae herein denotes the number of atoms in brackets to which the group is attached, for example in formulae (I),

n represents n

Attached to vicinal carbon atoms.

As used herein, the term "oligonucleotide chain" refers to an oligomeric compound containing multiple or all chemically modified or unmodified nucleotides, having a length of less than about 100 nucleotides (e.g., 1-20 nucleotides or 1-50 nucleotides). In certain embodiments, non-nucleic acid-conjugated groups may be included in the oligonucleotide chain. In certain embodiments, the oligonucleotide comprises ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or oligopeptide nucleotide (PNA). In certain embodiments, the oligonucleotide strand is double-stranded or single-stranded. In certain embodiments, the oligonucleotide strand is a siRNA, aptamer, antisense nucleic acid, sgRNA, tractRNA, or crRNA. In the sequences of the present application m before the nucleotide represents a 2'OMe modification and F represents a 2' F modification, e.g., mG represents glycine with a 2'OMe modification and fU represents uracil with a 2' F modification.

As used herein, "conjugate group" means an atom or group of atoms that is bound to the oligonucleotide chain. In certain instances, the conjugate groups alter one or more properties of the oligonucleotides to which they are attached, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and/or clearance properties.

As used herein, the term "conjugate" means a coupling molecule of a compound of the invention to an oligonucleotide chain. For example, a compound of formula (III) herein, and further for example, Z1001-Z1013 herein (wherein n, m and m' are each independently selected from integers between 1 and 10).

As used herein, the term "coupling reagent" means a compound or substance that can couple molecules of other compounds, for example, a compound of formula (I) or formula (II) herein, and further for example, GalNAc-1 to GalNAc-14 herein.

GalNAc-1

GalNAc-2

GalNAc-3

GalNAc-4

GalNAc-5

GalNAc-6

GalNAc-7：

GalNAc-8：

GalNAc-9：

GalNAc-10：

GalNAc-11：

GalNAc-12：

GalNAc-13

GalNAc-14

As used herein, the term "receptor" refers to a biological macromolecule composed of glycoproteins or lipoproteins, which are present in the cell membrane, cytoplasm, or nucleus, with different receptors having specific structures and configurations. As used herein, the term "ligand" refers to a substance or compound that has the ability to recognize and bind to a receptor. In certain embodiments, the ligand is a ligand that binds to asialoglycoprotein receptor (ASGPR). In certain embodiments, the ligands are carbohydrates, such as monosaccharides and polysaccharides, including but not limited to: galactose, N-acetylgalactosamine, mannose, glucose, glucosamine, and fucose.

As used herein, the term "polysaccharide" refers to a polymer made up of a plurality of monosaccharide groups joined glycosidically. In the present invention, the polysaccharide includes oligosaccharide and oligosaccharide. In general, "oligosaccharide" refers to a polymer in which 2 to 10 monosaccharide groups are linked together by glycosidic bonds, and "oligosaccharide" refers to a polymer in which 20 or less monosaccharide groups are linked together by glycosidic bonds.

As used herein, the term "about" should be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. If the meaning is not clear to a person skilled in the art from the context of the term application, "about" means a deviation of not more than plus or minus 10% of the stated particular value or range.

As used herein, the term "preventing" refers to preventing or delaying the onset of a disease.

As used herein, the term "treating" refers to curing or at least partially arresting the progression of a disease, or alleviating the symptoms of a disease.

As used herein, the term "effective amount" refers to an amount effective to achieve the intended purpose. For example, a disease-preventing effective amount is an amount effective to prevent, prevent or delay the onset of disease. It is within the ability of those skilled in the art to determine such an effective amount.

When a range of values is recited, it is intended to include each value and every subrange within the range. For example "C _1-6 Alkyl "includes C ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C _1-6 、C _1-5 、C _1-4 、C _1-3 、C _1-2 、C _2-6 、C _2-5 、C _2-4 、C _2-3 、C _3-6 、C _3-5 、C _3-4 、C _4-6 、C _4-5 And C _5-6 An alkyl group.

"alkyl" refers to a straight or branched chain saturated hydrocarbon group having carbon atoms. In some embodiments, C _1-6 Alkyl is preferred, C _1-4 Alkyl groups are particularly preferred. Examples of such alkyl groups include, but are not limited to: methyl (C) ₁ ) Ethyl (C) ₂ ) N-propyl (C) ₃ ) Isopropyl (C) ₃ ) N-butyl (C) ₄ ) Tert-butyl (C) ₄ ) Sec-butyl (C) ₄ ) Isobutyl (C) ₄ ) N-pentyl group (C) ₅ ) 3-pentyl radical (C) ₅ ) Pentyl group (C) ₅ ) Neopentyl (C) ₅ ) 3-methyl-2-butyl (C) ₅ ) Tert-amyl (C) ₅ ) And n-hexyl (C) ₆ ). Each of the alkyl groups is independently optionally substituted, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, whether or not the alkyl group is pre-modified with "substituted".

"alkenyl" refers to a straight or branched hydrocarbon group having at least one carbon-carbon double bond. In some embodiments, C _2-6 Alkenyl is preferred, C _2-4 Alkenyl groups are more preferred. C _2-6 Examples of alkenyl groups include: vinyl radical (C) ₂ ) 1-propenyl (C) ₃ ) 2-propenyl group (C) ₃ ) 1-butenyl (C) ₄ ) 2-butenyl (C) ₄ ) Butadienyl radical (C) ₄ ) Pentenyl (C) ₅ ) Pentadienyl (C) ₅ ) Hexenyl (C) ₆ ) And so on. The term "C _2-6 Alkenyl "also includes heteroalkenyl groups in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Each of the alkenyl groups is independently optionally substituted, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, regardless of whether the alkenyl group is modified with "substituted" or not.

"alkynyl" refers to a straight or branched hydrocarbon group having at least one carbon-carbon triple bond and optionally one or more carbon-carbon double bonds. In some embodiments, C _2-6 Alkynyl is preferred, C _2-4 Alkynyl groups are more preferred. C _2-6 Examples of alkynyl groups include, but are not limited to: ethynyl (C) ₂ ) 1-propynyl (C) ₃ ) 2-propynyl (C) ₃ ) 1-butynyl (C) ₄ ) 2-butynyl (C) ₄ ) Pentynyl group (C) ₅ ) Hexynyl (C) ₆ ) And so on. The term "C _2-6 Alkynyl also includes heteroalkynyl in which one or more (e.g., 1, 2, 3, or 4) carbon atoms are replaced with a heteroatom (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus). Each of the alkynyl groups is independently optionally substituted, whether or not the alkynyl group is modified by "substituted", for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, with appropriate substituents being defined below.

“C _6-14 Aryl "refers to a group having a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n +2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared in a cyclic arrangement) of 6 to 14 ring carbon atoms and zero heteroatoms. In some embodiments, an aryl group has six ring carbon atoms ("C) ₆ Aryl "; for example, phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C) ₁₀ Aryl "; e.g., naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms ("C) ₁₄ Aryl "; for example, an anthracene group). In some embodiments, C _6-10 Aryl is particularly preferred, more preferably C ₆ And (4) an aryl group. Aryl also includes ring systems in which the above-described aryl ring is fused to one or more cycloalkyl or heterocyclyl groups, and the point of attachment is on the aryl ring, in which case the number of carbon atoms continues to represent the number of carbon atoms in the aryl ring system. Each of the aryl groups is independently optionally substituted, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, whether or not the aryl group is modified "substituted" before the aryl group.

"3-to 10-membered heterocyclyl" is or refers to a group having a ring carbon atom and 1 to 4 ring heteroatoms in a 3-to 10-membered non-aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. In heterocyclic groups containing one or more nitrogen atoms, the point of attachment may be carbon or a nitrogen atom, as valency permits. In some embodiments, 3 to 7 membered heterocyclic groups are preferred, which are 3 to 7 membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; in some embodiments, 3 to 6 membered heterocyclic groups are particularly preferred, which are 3 to 6 membered non-aromatic ring systems having ring carbon atoms and 1 to 3 ring heteroatoms; more preferably a 5 to 6 membered heterocyclic group which is a 5 to 6 membered non aromatic ring system having ring carbon atoms and 1 to 3 ring heteroatoms. Heterocyclyl also includes ring systems wherein the aforementioned heterocyclyl ring is fused to one or more cycloalkyl, aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring; and in such cases the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Each of the heterocyclic groups is independently optionally substituted, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent, whether or not the heterocyclic group is modified "substituted" before the heterocyclic group.

Exemplary 3-membered heterocyclic groups containing one heteroatom include, but are not limited to: aziridinyl, oxacyclopropaneyl, thienylyl. Exemplary 4-membered heterocyclic groups containing one heteroatom include, but are not limited to: azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to: tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2, 5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to: dioxolanyl, oxathiolanyl (oxathiolanyl), dithiolanyl (disulphenyl) and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing three heteroatoms include, but are not limited to: triazolinyl, oxadiazolinyl and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing one heteroatom include, but are not limited to: piperidinyl, tetrahydropyranyl, dihydropyridinyl and thiacyclohexyl (thianyl). Exemplary 6-membered containing two heteroatomsHeterocyclic groups include, but are not limited to: piperazinyl, morpholinyl, dithiinyl, dioxanyl. Exemplary 6-membered heterocyclic groups containing three heteroatoms include, but are not limited to: hexahydrotriazinyl (triazinanyl). Exemplary 7-membered heterocyclic groups containing one heteroatom include, but are not limited to: azepane, oxepanyl and thiepane. Exemplary 8-membered heterocyclic groups containing one heteroatom include, but are not limited to: azacyclooctyl, oxocyclooctyl and thietanyl. Exemplary with C ₆ Aryl ring fused 5-membered heterocyclyl (also referred to herein as 5, 6-bicyclic heterocyclyl) includes, but is not limited to: indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolonyl, and the like. Exemplary with C ₆ Aryl ring fused 6-membered heterocyclyl (also referred to herein as 6, 6-bicyclic heterocyclyl) includes, but is not limited to: tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

Exemplary substituents include, but are not limited to: halogen, -CN, -NO ₂ 、-N ₃ 、-SO ₂ H、-SO ₃ H. -OH, alkyl, haloalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl and heteroaryl.

The term "stereoisomers" refers to compounds having the same chemical structure, but differing in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis/trans), atropisomers, and the like.

The stereochemical definitions and rules used in the present invention generally follow the general definitions of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E.and Wilen, S, "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc, New York, 1994. Many organic compounds exist in an optically active form, i.e., they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of a molecule with respect to one or more of its chiral centers. The prefixes d and l or (+) and (-) are the symbols used to specify the rotation of plane polarized light by the compound, where (-) or l indicates that the compound is left-handed. Compounds prefixed with (+) or d are dextrorotatory. A particular stereoisomer is an enantiomer and a mixture of such isomers is referred to as an enantiomeric mixture. A50: 50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in the chemical reaction or process.

Any resulting mixture of stereoisomers may be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, depending on differences in the physicochemical properties of the components, for example, by chromatography and/or fractional crystallization.

The term "tautomer" or "tautomeric form" refers to structural isomers having different energies that can interconvert by a low energy barrier (low energy barrier). If tautomerism is possible (e.g., in solution), then the chemical equilibrium of the tautomer can be reached. For example, proton tautomers (also known as proton transfer tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization.

The term "pharmaceutically acceptable salts" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, the pharmaceutically acceptable salts are described in detail by Berge et al in J.pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Salts formed using methods conventional in the art, e.g., ion exchange methods, are also included.Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cypionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, picrate, etc, Stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, pentanoates, and the like. Pharmaceutically acceptable salts derived from suitable bases include alkali metals, alkaline earth metals, ammonium and N ⁺ (C _1-4 Alkyl radical) ₄ And (3) salt. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. Other pharmaceutically acceptable salts include, if appropriate, non-toxic ammonium, quaternary ammonium and amine cations formed with counterions such as halide, hydroxide, formate, sulfate, phosphate, nitrate, lower alkyl sulfonate and aryl sulfonate.

Treatment of

The "subject" to which the drug is administered includes, but is not limited to: a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, an adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an older adult)) and/or a non-human animal, e.g., a mammal, e.g., a primate (e.g., a cynomolgus monkey, a rhesus monkey), a cow, a pig, a horse, a sheep, a goat, a rodent, a cat, and/or a dog. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human", "patient" and "subject" are used interchangeably herein.

In the methods of treatment of the present invention, an "effective amount" refers to an amount or dose sufficient to produce the desired therapeutic benefit in an individual in need of such treatment. An effective amount or dose of a compound of the invention can be determined by conventional methods (e.g., modeling, dose escalation, or clinical trials) and by conventional factors (e.g., the mode or route of drug delivery, the pharmacokinetics of the agent, the severity and course of the infection, the health and weight of the individual, and the judgment of the treating physician). Exemplary doses are in the range of about 0.1mg to 1g per day, or about 1mg to 50mg per day, or about 50mg to 250mg per day, or about 250mg to 1g per day. The total dose can be administered as a single dose or as separate dosage units (e.g., BID, TID, QID).

After the patient has developed an improvement in the disease, the dosage can be adjusted for prophylactic or maintenance treatment. For example, the dosage or frequency of administration, or both, can be reduced to an amount that maintains the desired therapeutic or prophylactic effect, depending on the symptoms. Of course, if the symptoms have been alleviated to an appropriate degree, treatment may be discontinued. However, when either symptom recurs, the patient may require long-term intermittent treatment. Patients may also require chronic treatment for extended periods of time.

Pharmaceutical compositions, formulations and kits

The present invention provides pharmaceutical compositions comprising a compound or conjugate of the invention (also referred to as "active ingredient") and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises an effective amount of an active ingredient. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an active ingredient. In some embodiments, the pharmaceutical composition comprises a prophylactically effective amount of an active ingredient.

Pharmaceutically acceptable excipients for use in the present invention refer to non-toxic carriers, adjuvants or vehicles that do not destroy the pharmacological activity of the compounds or conjugates formulated therewith. Pharmaceutically acceptable carriers, adjuvants, or vehicles that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

The invention also includes kits (e.g., pharmaceutical packages). The provided kits can include a compound of the invention, an additional therapeutic agent, and first and second containers (e.g., vials, ampoules, bottles, syringes, and/or dispensable packages or other suitable containers) containing the compound of the invention, the additional therapeutic agent. In some embodiments, provided kits may also optionally include a third container containing a pharmaceutically acceptable excipient for diluting or suspending a compound of the invention and/or other therapeutic agent. In some embodiments, the compound of the present invention and the additional therapeutic agent provided in the first container and the second container are combined to form one unit dosage form.

The pharmaceutical compositions provided by the present invention may be administered by a number of routes including, but not limited to: oral, parenteral, inhalation, topical, rectal, nasal, buccal, vaginal, by implant or other modes of administration. For example, parenteral administration as used herein includes subcutaneous administration, intradermal administration, intravenous administration, intramuscular administration, intraarticular administration, intraarterial administration, intrasynovial administration, intrasternal administration, intracerebrospinal administration, intralesional administration, and intracranial injection or infusion techniques.

Typically, an effective amount of a compound provided herein is administered. The amount of compound actually administered can be determined by a physician, as the case may be, including the condition to be treated, the chosen route of administration, the compound actually administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.

When used to prevent a condition according to the invention, a subject at risk of developing the condition is administered a compound provided herein, typically based on physician's advice and under the supervision of a physician, at a dosage level as described above. Subjects at risk of developing a particular disorder, typically include subjects with a family history of the disorder, or those determined to be particularly susceptible to developing the disorder by genetic testing or screening.

The pharmaceutical compositions provided herein may also be administered chronically ("chronic administration"). By long-term administration is meant administration of the compound or pharmaceutical composition thereof over a long period of time, e.g., 3 months, 6 months, 1 year, 2 years, 3 years, 5 years, etc., or may continue for an indefinite period of time, e.g., for the remainder of the subject's life. In some embodiments, chronic administration is intended to provide a constant level of the compound in the blood over a prolonged period of time, e.g., within the therapeutic window.

Various methods of administration may be used to further deliver the pharmaceutical compositions of the present invention. For example, in some embodiments, the pharmaceutical composition may be administered as a bolus, e.g., in order to rapidly increase the concentration of the compound in the blood to an effective level. The bolus dose depends on the targeted systemic level of the active ingredient, e.g., an intramuscular or subcutaneous bolus dose results in a slow release of the active ingredient, while a bolus delivered directly to the vein (e.g., by IV intravenous drip) can be delivered more rapidly, allowing the concentration of the active ingredient in the blood to rise rapidly to an effective level. In other embodiments, the pharmaceutical composition may be administered as a continuous infusion, e.g., by IV intravenous drip, to provide a steady state concentration of the active ingredient in the body of the subject. Furthermore, in other embodiments, a bolus dose of the pharmaceutical composition may be administered first, followed by continuous infusion.

In order to provide blood levels similar to, or lower than, those used with the injected dose, transdermal doses are generally selected in amounts of from about 0.01 to about 20% by weight, preferably from about 0.1 to about 10% by weight, and more preferably from about 0.5 to about 15% by weight.

From about 1 to about 120 hours, especially 24 to 96 hours, the injection dosage level is in the range of about 0.1 mg/kg/hour to at least 10 mg/kg/hour. To obtain sufficient steady state levels, a preload bolus of about 0.1mg/kg to about 10mg/kg or more may also be administered. For human patients of 40 to 80kg, the maximum total dose cannot exceed about 2 g/day.

Injectable compositions are typically based on sterile saline or phosphate buffered saline for injection, or other injectable excipients known in the art. As previously mentioned, in such compositions, the active compound is typically a minor component, often about 0.05 to 10% by weight, with the remainder being injectable excipients and the like.

The above components of the compositions for injection or topical administration are merely representative. Other materials and processing techniques are described in Remington's Pharmaceutical Sciences,17th edition,1985, Mack Publishing Company, Easton, Pennsylvania, section 8, which is incorporated herein by reference.

The compounds of the present invention may also be administered in sustained release form, or from a sustained release delivery system. A description of representative sustained release materials can be found in Remington's Pharmaceutical Sciences.

The invention also relates to pharmaceutically acceptable formulations of the compounds of the invention. In one embodiment, the formulation comprises water.

Pharmaceutical composition

The compounds or conjugates of the invention described herein may be used in combination with one or more other active ingredients in pharmaceutical compositions or methods for the treatment of diseases and conditions described herein. Other additional active ingredients include other therapeutic agents or agents that mitigate the adverse effects of treatment against the intended disease target. The combinations can be used to increase efficacy, ameliorate other disease symptoms, reduce one or more side effects, or reduce the required dose of the compounds of the invention. The additional active ingredients may be formulated as separate pharmaceutical compositions from the compounds of the present invention or may be included in a single pharmaceutical composition with the compounds of the present invention. The additional active ingredient may be administered simultaneously with, before or after the administration of the compound of the invention.

Combination agents include those active ingredients known or observed to be effective in treating the diseases and conditions described herein, including those effective against another target associated with the disease. For example, the compositions and formulations of the invention, as well as methods of treatment, may further comprise other drugs, such as other agents useful for treating or ameliorating a target disease or associated symptoms or conditions. The pharmaceutical compositions of the invention may additionally comprise one or more of said active agents, and the method of treatment may additionally comprise administering an effective amount of one or more of said active agents.

Embodiments of the present invention will be described in further detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 preparation of GalNAc-1

1. Synthetic route

2. Synthesis method

(1) Dissolving 50g of pentaerythritol in 150g of tert-butyl acrylate, adding 6ml of 50% sodium hydroxide solution, adding 9.5g of tetrabutylammonium hydroxide, reacting at room temperature completely, adding ethyl acetate, extracting to obtain an organic phase, concentrating to dryness, and separating and purifying by a column to obtain a compound 1(PE (petroleum ether): EA (ethyl acetate): 20: 1-7: 1); (2) 10g of Compound 1 and 6.5g of TsCl were dissolved in 100ml of Py (pyridine) solution, and the reaction was stirred at 70 ℃. After the reaction is completed, concentrating to remove pyridine, adding ethyl acetate to dilute, drying, concentrating, and performing column separation and purification to obtain a compound 2 (PE: EA is 20: 1-10: 1); (3) dissolving 4.2g of compound 2 in 50ml of anhydrous DMF, adding 0.53g of sodium azide, stirring at 100 ℃ to react completely, concentrating to remove DMF, adding ethyl acetate for liquid separation and extraction, concentrating to dryness, and separating and purifying by a column to obtain a compound 3 (PE: EA is 20: 1-10: 1); (4) dissolving 2g of the compound 3 in 10ml of formic acid, stirring to react completely, and concentrating to dryness to obtain a compound 4; (5) dissolving 1.6g of compound 4 in 50ml of dichloromethane, adding 2.23g of HOBt, 3.0g of EDCI, 3.3g of DIEA and 2.7g of mono-Boc-propylenediamine, stirring at room temperature until the reaction is completed, adding dichloromethane for extraction, drying and concentrating, and separating and purifying by a column to obtain a compound 5 (dichloromethane: methanol is 2% -5%); (6) dissolving 2.5g of the compound 5 in 50ml of dichloromethane, adding 20ml of 2N trifluoroacetic acid, reacting completely at room temperature, concentrating and draining to obtain a compound 6; (7) dissolving 11g of galactovaleric acid in 100ml of dichloromethane, adding 6.2g of DCC and 5.5g of pentafluorophenol, stirring at room temperature until the reaction is completed, filtering, washing an organic phase with water, drying, concentrating to dryness, and separating and purifying by a column to obtain a compound 7 (PE: EA is 3: 1-1: 1); (8) dissolving 2.46g of compound 6 and 10g of compound 7 in 50ml of dichloromethane solution, adding 8ml of DIEA, stirring at room temperature until the reaction is completed, adding dichloromethane for extraction, drying and concentrating the organic phase, and separating and purifying by a column to obtain a compound 8 (dichloromethane: methanol is 10% -20%); (9) dissolving 10g of glutaric anhydride and 4.82g of propargylamine in 50ml of tetrahydrofuran, stirring at room temperature for complete reaction, and concentrating to dryness to obtain a compound 12; (10) dissolving 3.7g of the compound 12 in 50ml of dichloromethane, adding 5.41g of DCC and 4.83g of pentafluorophenol, stirring at room temperature until the reaction is completed, filtering, washing an organic phase with water, drying, concentrating to dryness, and separating and purifying by a column to obtain a product, namely a compound 13 (PE: EA is 3: 1-1: 1); (11) dissolving 6.5g of compound 13 and 2.54g of aminocaproic acid in 50ml of tetrahydrofuran, stirring at room temperature until the reaction is completed, concentrating to be dry, adding dichloromethane to extract an organic phase, drying, concentrating to be dry, and separating and purifying by a column to obtain compound 14 (dichloromethane: methanol is 3% -8%); (12) dissolving 4.5g of the compound 14 in 100ml of dichloromethane, adding 3.94g of DCC and 3.52g of pentafluorophenol, stirring at room temperature until the reaction is completed, filtering, washing an organic phase with water, drying, concentrating to dryness, and separating and purifying by a column to obtain a product compound 15 (PE: EA is 3: 1-1: 1); (13) 0.34g of the compound 8 was dissolved in 5ml of THF, and 5ml of an aqueous solution of 0.09g of the compound 15, 0.092g of anhydrous copper sulfate and 0.146g of sodium ascorbate was added thereto, and the reaction mixture was stirred at room temperature and then completely reacted, followed by removal of THF by concentration, dilution with methylene chloride, drying and decoloring with activated carbon and anhydrous sodium sulfate, removal of insoluble matter by filtration, concentration to dryness, and purification by column chromatography to obtain GalNAc-1 (methylene chloride: methanol: 10% to 15%).

3. Results of the experiment

About 0.4g GalNAc-1 was obtained. White foamy solid.

¹ HNMR(400MHz,CDCl ₃ )δ:ppm.7.69(s,1H),7.36,dd,4H),6.97(t,3H),6.81(d,1H)，6.74(d，2H),6.40(s，1H)，5.35(t,3H)，5.19(dd，3H),4.61(d，3H)，4.51(d，2H),4.32(s,2H)，4.13(m，9H)，3.92(dd，6H)，3.65(d，6H)，3.50(m，3H)，3.27(m，18H)，2.68(t，2H)，2.44(t，6H)，2.33(t，2H)，2.21-1.26(m，61H)。MS(ESI-TOF):m/z(M+H) ⁺ 2283.44，(M+Na) ⁺ 2305.41。

EXAMPLE 2 GalNAc-2 preparation

1. Synthetic route

The synthesis methods of compound 8 and compound 13 are the same as in example 1.

2. The synthesis method comprises the following steps: prepared according to the synthetic method of reference example 1.

3. Results of the experiment

About 0.2g GalNAc-2 was obtained. White foamy solid.

1HNMR(400MHz，CDCl3)δ:ppm 7.73(s，1H),6.90-6.40(m,10H),5.36(d,3H),5.08(m,3H),4.60(s,3H),4.50(d,2H),4.35(s,2H),4.14(m,9H),3.92(s,6H),3.66(s,6H),3.51(m,3H),3.27(m,18H),2.79(t,2H),2.45(m,6H),2.25-1.80(m,44H),1.80-1.26(m,20H)

MS(ESI-TOF):m/z(M+Na) ⁺ 2191.38。

Example 3 preparation of GalNAc-3

1 synthetic route

The synthesis methods of compound 4 and compound 15 were the same as in example 1.

2. Synthesis method

(1) With reference to the synthesis methods of Compound 7, Compound 8 and GalNAc-1 in example 1, Compound 9, Compound 11 and GalNAc-3 were obtained;

(2) 4.5g of the compound Cbz-hexylamine-galactose (purchased from Alading) is dissolved in 100ml of ethyl acetate, 1g of 10% palladium carbon is added, and after complete hydrogenation reaction under stirring at room temperature, the product compound 10 is obtained by filtering, drying and concentrating to dryness.

3. Results of the experiment

About 0.2g of GalNAc-3 was obtained. White foamy solid.

¹ HNMR(400MHz,CDCl ₃ )δ：ppm.7.47-6.32(m,9H),5.36(s,3H),5.28(m,3H),4.69(s,3H),4.54(s,2H),4.36(m,2H),4.15(m,6H),4.05(m,3H),3.94(dd,6H),3.66(s,6H),3.47(m,3H),3.25(m,12H),2.68(t,2H),2.44(m,6H),2.30-1.80(m,42H),1.80-1.26(m,32H).

MS(ESI-TOF):m/z(M+Na) ⁺ 2133.43。

Example 4 preparation of GalNAc-4

1. Synthetic route

The synthesis of compound 13 was performed as in example 1, and the synthesis of compound 11 was performed as in example 3.

3. Results of the experiment

About 0.2g GalNAc-4 was obtained. White foamy solid.

¹ HNMR(400MHz,CDCl ₃ )δ：ppm.7.74-6.43(m,8H)，5.36(d，3H)，5.28(m,3H),4.68(d,3H)，4.53(d，2H)，4.31(s，2H),4.14(ddd,6H),4.05(dd,3H),3.89(ddd,6H),3.67(m,6H),3.46(dd,3H),3.24(s,12H),2.78(t,2H),2.43(m,6H),2.30-1.80(m,38H),1.80-1.26(m,26H).

MS(ESI-TOF)：m/z(M+H) ⁺ 1998.43,(M+Na) ⁺ 2020.42.

Example 5 preparation of GalNAc-5

1. Synthetic route

The synthesis of compound 11 was performed as in example 3.

2. Synthesis method

(1) Dissolving 1.6g of compound 16 in 50ml of dichloromethane, adding 2.23g of HOBt, 3.0g of EDCI, 3.3g of DIEA and 0.3g of propargylamine, stirring at room temperature until the reaction is completed, adding dichloromethane to extract an organic phase, drying and concentrating, and separating and purifying by a column to obtain compound 17 (dichloromethane: methanol is 2% -5%); (2) dissolving 2g of raw materials in 10ml of methanol, adding 0.5g of palladium carbon (10%), completely hydrogenating, filtering, collecting filtrate, and concentrating to dryness to obtain a compound 18; (3) dissolving 4.5g of PFP-dodecanoate in 100ml of dichloromethane, adding 3.94g of DCC and 3.52g of pentafluorophenol, stirring at room temperature for complete reaction, filtering, drying, concentrating to dryness, and performing column separation and purification (PE: EA is 3: 1-1: 1) to obtain a compound 19; (4) referring to the synthesis method of GalNAc-1 in example 1, GalNAc-5 was obtained.

3. Results of the experiment

About 0.4g GalNAc-5 was obtained. White foamy solid.

¹ HNMR(400MHz,CDCl ₃ )δ：ppm。7.73-6.40(m,8H),5.35(d,3H),5.26(m,3H),4.67(d,3H)，4.51(s,2H),4.33(s,2H),4.11(dd,6H),4.07(d,3H),3.89(dt,6H),3.66(m,6H),3.46(dd,3H),3.24(s,12H),2.75(t,2H),2.41(m,6H)，2.25-1.80(m,38H),1.80-1.26(m,40H).

MS(ESI-TOF)：m/z(M+H) ⁺ 2096.19,(M+Na) ⁺ 2119.20.

Example 6 preparation of GalNAc-6

1. Synthetic route

The synthesis of compound 8 was performed as in example 1.

2. Synthesis method

(1) Dissolving 47.9g of aminocaproic acid in 700ml of toluene, adding 57ml of benzyl alcohol and 74g of 4-toluenesulfonic acid, stirring at 115 ℃ until the reaction is complete, cooling to room temperature, stirring until a large amount of solid is separated out, adding 500ml of methyl tert-butyl ether, filtering, pumping and drying to obtain a compound 22; (2) dissolving 19g of the compound 22 in 100ml of dichloromethane, adding 11g of glutaric anhydride in 100ml of dichloromethane solution, stirring at room temperature for complete reaction, and drying and concentrating to obtain a compound 23; (3) dissolving 5g of the compound 23 in 100ml of dichloromethane, adding 3.69g of DCC and 3.3g of pentafluorophenol, stirring at room temperature until the reaction is completed, filtering, concentrating to dryness, and separating and purifying by a column to obtain a compound 24 (PE: EA is 3: 1-2: 1); (4) dissolving 0.13g of the compound 8 in 10ml of ethyl acetate and 10ml of anhydrous methanol, adding 0.2g of palladium carbon and 3 drops of acetic acid, performing displacement reaction in hydrogen, filtering, and concentrating the filtrate to dryness to obtain a compound 25; (5) dissolving 2.8g of the compound 25 in 50ml of dichloromethane, adding 0.5g of DIEA and 0.78g of the compound 24, reacting completely at room temperature, drying and concentrating to dryness, and separating and purifying by a column to obtain a compound 26 (dichloromethane: 15% -25% of methanol); (6) dissolving 2.5g of the compound 26 in 30ml of methanol and 30ml of ethyl acetate, adding 1.3g of palladium carbon to perform hydrogen replacement reaction, filtering, drying and concentrating the filtrate to dryness to obtain a compound 27; (7) 0.8g of the compound 27 was dissolved in dichloromethane, 0.12g of DCC and 0.11g of pentafluorophenol were added, after completion of the reaction at room temperature, dichloromethane was added to dilute the reaction solution, and the solution was dried and concentrated to dryness, followed by column separation and purification to obtain GalNAc-6 (dichloromethane: methanol 10% -15%).

3. Results of the experiment

About 0.7g GalNAc-6 was obtained. White foamy solid.

¹ HNMR(400MHz,d-DMSO)δ：ppm.7.75-7.26(m,11H),5.85(d，3H),5.59(d，3H)，5.11(dd，3H),4.63(m,6H)，4.50(d,6H),4.37(s,3H),4.17(d,6H),4.07(s,3H),3.90-3.70(m,18H),3.28(m,2H),2.92(d,6H),2.80-2.25(m,52H),2.25-1.26(m,24H).

MS(ESI-TOF)：m/z(M+H) ⁺ 2201.82，(M+Na) ⁺ 2223.86。

Example 7 preparation of GalNAc-7

1. Synthetic route

The synthesis of compound 25 was performed as in example 6.

2. Synthesis method

(1) Reference example 1 synthesis of compound 13 gave compound 21; (2) with reference to the synthesis methods of Compound 26, Compound 27 and GalNAc-6 in example 6, Compound 29, Compound 30 and GalNAc-7 were obtained.

3. Results of the experiment

About 0.5g GalNAc-7 was obtained. White foamy solid.

¹ HNMR(400MHz,CDCl ₃ )δ：ppm 7.32-6.63(m,10H),5.35(s,3H),5.20(s,3H),4.62(d，3H)，4.13(ddd，9H),3.92(d，6H),3.66(dd，6H)，3.52(d，3H),3.33(m，20H)，2.77(t，2H),2.44(s，6H)，2.25-1.80(m，44H),1.80-1.26(m，20H).

MS(ESI-TOF)：m/z(M+H) ⁺ 2088.08，(M+Na) ⁺ 2109.36。

Example 8 preparation of GalNAc-8

1. Synthetic route

The synthesis of compound 11 was performed as in example 3, and the synthesis of compound 24 was performed as in example 6.

2. The synthesis method comprises the following steps: with reference to the synthesis methods of Compound 25, Compound 26, Compound 27, GalNAc-6 in example 6, Compound 32, Compound 36, Compound 37, GalNAc-8 were obtained;

3. results of the experiment

About 0.45g GalNAc-8 was obtained.

¹ HNMR(400MHz,CDCl ₃ )δ：ppm.7.7.06-6.37(m,8H),5.36(d,3H),5.270(m,3H),4.69(t,3H),4.15(m,6H),4.00(m,3H),3.90(m,6H),3.65(t,6H),3.47(dt,3H),3.28(m,16H),2.68(t,2H),2.42(m,6H),2.29(t,4H),2.15-1.80(s,36H),1.80-1.26(m,32H)。

MS(ESI-TOF)：m/z(M+H) ⁺ 2030.38，(M+Na) ⁺ 2052.41。

Example 9 preparation of GalNAc-9

1. Synthetic route

The synthesis of compound 11 was performed as in example 3.

2. Synthesis method

(1)1.3g of azide compound 11 is dissolved in 15ml of THF, 0.3g of palladium carbon and 0.11g of glutaric anhydride are added, hydrogen substitution reaction is carried out, after the reaction is completed at room temperature, the palladium carbon is removed by filtration, the filtrate is concentrated to be dry, dichloromethane is added to dilute and extract an organic phase, and concentration and column separation purification are carried out (dichloromethane: methanol is 10% -15%), so that about 0.8g of product compound 34 is obtained.

(2) Referring to the synthesis method of GalNAc-6 in example 6, GalNAc-9 was obtained.

3. Results of the experiment

About 0.8g GalNAc-9 was obtained. A foamy solid.

¹ HNMR(400MHz,d-DMSO)δ：ppm.7.63(s,1H),7.42(s，3H)，7.17(s,3H)，5.85(d,3H)，5.60(t,3H),5.10(dd,3H),4.63(td,6H),4.49(m,6H),4.34(d,3H),4.16(m,6H),4.03(d,3H),3.84(d,6H),3.73(d,6H),3.35(m,2H),2.91(d,6H),2.70-2.40(m,42H),2.21-1.76(m,24H)。

MS(ESI-TOF)：m/z(M+H) ⁺ 1917.93，(M+Na) ⁺ 1938.69。

Example 10 preparation of GalNAc-10

1 synthetic route

Method for synthesis of compound 28 reference was made to the synthesis of compound 24 in example 6.

2. Synthesis method

(1) Dissolving 3g of amino compound in 30ml of dichloromethane, adding DIEA (1ml) and 1.31g of monobenzyldodecanoic acid-pentafluorophenol ester, reacting completely at room temperature, adding dichloromethane for dilution, drying and concentrating to dryness, and separating and purifying by a column (dichloromethane: methanol is 10% -15%) to obtain a compound 38; (2) referring to the synthesis method of compound 27, GalNAc-6 in example 6, compound 39, GalNAc-10 was obtained.

3. Results of the experiment

About 0.45g GalNAc-10 was obtained.

¹ HNMR(400MHz,CDCl ₃ )δ：ppm 7.7.06-6.40(m,7H),5.35(d,3H),5.28(m,3H),4.67(t,3H)，4.14(m,6H),4.02(m,3H),3.93(m,6H),3.61(t,6H),3.48(dt,3H),3.29(m,14H),2.67(t,2H),2.43(m,6H),2.28(t,2H),2.15-1.80(s,36H),1.80-1.26(m,40H)。

MS(ESI-TOF)：m/z(M+H) ⁺ 2015.11,(M+Na) ⁺ 2038.47。

Example 11 preparation of GalNAc-11

1. Synthetic route

The synthesis of the compound GalNAc-4 was performed in the same manner as in example 4.

2. Synthesis method

(1) Dissolving 3g of GalNAc-4 in 30ml of dichloromethane, adding DIEA (0.3ml) and 0.18g of 1-hydroxycaproic acid, completely reacting at room temperature, diluting the dichloromethane, drying and concentrating to dryness, and separating and purifying by a column (dichloromethane: methanol is 10% -15%) to obtain a compound 40; (2)1g of the compound 40 was dissolved in 10ml of anhydrous dichloromethane, DIEA (0.2ml) and phosphorous acid chloride (0.14g) were added thereto, and the mixture was stirred at 0 ℃ to react completely, diluted with dichloromethane, dried and concentrated, and purified by column chromatography (dichloromethane: methanol 10% -15%) to obtain GalNAc-11.

3. Results of the experiment

The spectral data for compound 40 was:

¹ HNMR(400MHz,CDCl3)δ：ppm.7.71-6.38(m,9H),5.36(d,3H),5.27(m,3H),4.68(d,3H),4.53(s,2H),4.31(s,2H),4.15(m,6H),4.01(dd,3H)),3.89(ddd,6H),3.65(dd,8H),3.46(m，3H)，3.24(m，14H)，2.46(d，6H)，2.31(d，2H)，2.23(s，2H)，2.16-1.80(m,36H),1.80-1.26(m,34H)。

MS(ESI-TOF)：m/z(M-H) ^- 1929.81.

about 0.5g GalNAc-11 was obtained. White foamy solid. MS (ESI-TOF): m/z (M + Na) ⁺ 2153.62。

Example 12 preparation of GalNAc-12

1. Synthetic route

Compound 42 (purchased from Alading), compound GalNAc-5 is the same as in example 5.

2. Synthesis method

(1) Dissolving 2g of GalNAc-5 in 20ml of dichloromethane, adding DIEA (0.3ml) and DMTr-hydroxyproline (compound 42)0.44g, completely reacting at room temperature, diluting with dichloromethane, drying, concentrating to dryness, and separating and purifying by a column (dichloromethane: methanol 10% -15%) to obtain a compound 41; (2)1g of the compound 41 was dissolved in 10ml of anhydrous dichloromethane, DIEA (0.2ml) and phosphorous acid chloride (0.12g) were added thereto, and after completion of the reaction with stirring at 0 ℃ the mixture was diluted with dichloromethane, dried and concentrated, and then subjected to column separation and purification (dichloromethane: methanol 10% to 15%) to obtain GalNAc-12.

3. Results of the experiment

About 0.6g GalNAc-12 was obtained. White foamy solid.

EXAMPLE 13 preparation of modified Single-stranded oligonucleotide (antisense strand)

Examples 13, 16-18 relate to the following exemplary antisense strand sequences:

sequence 1(SEQ ID NO: 1):

5'-Cy5-mUmGmAfCmAmAmAfCmGmGmGfCmAmAfCmAfUmAfC-3'

in this example, modified oligonucleotides were synthesized according to the theoretical yield 1 μmol specification, as follows:

(1) mu. mol of standard general solid support CPG (controlled pore glass) or 3 '-cholesterol modified CPG (purchased from Chemmenes), or 3' -amino modified-solid support (purchased from Kinovite),2 '-O-TBDMS RNA phosphoramidite protected monomer, DNA monomer, 2' -methoxy monomer, 2 '-fluoro monomer (purchased from Sigma Aldrich), phosphoramidite used for synthesizing 5' -modified amino-C16-12-phosphoramidite or fluorescent compound was dissolved in anhydrous acetonitrile to a concentration of 0.2M. For the phosphate backbone thio-modified oligonucleotides, 0.2M PADS solution was used as the thioreagent. 5-ethylthio-1H-tetrazole (purchased from Chemchromenes) acetonitrile solution is prepared as an activating agent (0.25M), 0.02M iodine pyridine/water solution is prepared as an oxidizing agent, and 3% trichloroacetic acid dichloromethane solution is prepared as a deprotection reagent, and the reagents are placed at the corresponding reagent designated positions of a DNA/RNA automatic synthesizer (GE AKTAOP 100).

(2) Setting a synthesis program, inputting a specified oligonucleotide base sequence, checking to be correct, starting to synthesize cyclic oligonucleotides, wherein the coupling time of each step is 6 minutes, and the coupling time of the galactose ligand corresponding to the monomer is 10-20 minutes. After automatic circulation, the oligonucleotide solid phase synthesis is completed.

(3) The CPG was blown dry with dry nitrogen, transferred to a 5mL EP tube, added with 2mL of ammonia/ethanol solution (3/1), and heated at 55 ℃ for 16-18 hours. Centrifuging at 10000rpm for 10min to obtain supernatant, and draining off concentrated ammonia water/ethanol to obtain white colloidal solid. The solid was dissolved in 200. mu.L of 1M TBAF THF and shaken at room temperature for 20 hours. 0.5mL of 1M Tris-HCl buffer (pH 7.4) was added, shaken at room temperature for 15 minutes, and then placed in a centrifugal pump to pump the mixture to 1/2, the volume of which was the original volume, and THF was removed. The solution was extracted 2 times with 0.5mL of chloroform, 1mL of 0.1M TEAA loading was added, the mixture was poured into a solid phase extraction column, and excess salt was removed from the solution.

(4) The concentration of the obtained oligonucleotide was measured by a micro ultraviolet spectrophotometer (KO 5500). Mass spectrometric detection analysis was performed on an Oligo HTCS LC-MS system (Novatia). Nucleic acid molecular weights were calculated by normalization with Promass software after the primary scan.

Example 14 preparation of modified Single-stranded oligonucleotide (sense Strand)

Examples 14-18 refer to exemplary sense strand sequences as follows:

sequence 2(SEQ ID NO: 2):

5'-mGfUmAfUmGfUfUmGfCfCfCmGfUfUfUmGfUfCmA-3'

in this example, modified oligonucleotides were synthesized according to the theoretical yield 1. mu. mol specification as follows:

(1) mu. mol of CPG or 3 ' -cholesterol-modified CPG (purchased from Chemmenes), 3 ' -amino-modified solid phase support (purchased from Kinovite), DNA monomer, 2' -methoxy monomer, 2' -fluoro monomer (purchased from Sigma Aldrich), or phosphoramidite used for synthesizing 5 ' -modified amino-C16-12-phosphoramidite or fluorescent compound was weighed and dissolved in anhydrous acetonitrile to a concentration of 0.2M. For the phosphate backbone thio-modified oligonucleotides, 0.2M PADS solution was used as the thioreagent. 5-ethylthio-1H-tetrazole (purchased from Chemgenes) acetonitrile solution is prepared as an activating agent (0.25M), 0.02M iodine pyridine/water solution is prepared as an oxidizing agent, and 3% trichloroacetic acid dichloromethane solution is prepared as a deprotection reagent and is placed at a corresponding reagent designated position of a DNA/RNA automatic synthesizer.

(2) Setting a synthesis program, inputting a specified oligonucleotide base sequence, checking to be correct, starting to synthesize cyclic oligonucleotides, wherein the coupling time of each step is 6 minutes, and the coupling time of the galactose ligand corresponding to the monomer is 6-10 minutes. After automatic circulation, the oligonucleotide solid phase synthesis is completed.

(3) And drying the CPG by using dry nitrogen, transferring the CPG into a 5mL EP tube, adding 2mL of ammonia solution, and heating at 55 ℃ for 16-18 hours. Centrifuging at 10000rpm for 10min to obtain supernatant, and draining off concentrated ammonia water/ethanol to obtain white or yellow colloidal solid. 1mL of 0.1M TEAA loading solution was added, and the mixed solution was poured into a solid phase extraction column to remove excess salt from the solution.

EXAMPLE 15 preparation of GalNAc-modified oligonucleotides

Amino-modified oligonucleotides prepared according to the method of example 14 were dissolved in a buffer, various GalNAc-pentafluorophenol esters (GalNAc is selected from GalNAc-1 to GalNAc-10) dissolved in acetonitrile solvent were added to the amino-modified oligonucleotide solution, mixed well, reacted at room temperature for at least 3 hours, the acetyl protecting group was removed after the reaction was completed, the GalNAc-modified oligonucleotides were purified by ion exchange chromatography (WATERS) with DNAPAc PA-100 ion exchange column linear gradient, mobile phase a: 20mM NaOH; mobile phase B: 20mM NaOH +2M NaCl mixture. The sequences of exemplary GalNAc modified oligonucleotides and the corresponding molecular weight measurements are shown in table 1.

TABLE 1 GalNAc-modified oligonucleotides (sense strand)

Description of abbreviations: n ═ RNA; dN ═ DNA; mN ═ 2' OMe modification; fN-2' F modification.

EXAMPLE 16 preparation of double-stranded oligonucleotide

The process is as follows: the 5' -Cy 5-antisense strand oligonucleotide synthesized in example 13 was compared with the GalNAc-1-10 sense strand oligonucleotide in example 15, respectively, in terms of UV absorption content 1: 1 mixing, heating to 95 deg.C in a water bath for three minutes, cooling to room temperature to form GalNAc-double chain (shown in Table 2)

TABLE 2 GalNAc-double stranded RNA Structure

Example 17 cell-targeting assay of modified oligonucleotides

Modified oligonucleotides for animal experiments were filtered through 0.22 μm membranes before injection.

1. Isolation of mouse primary hepatocytes

Mice (purchased from beijing weitonglihua laboratory animals ltd.) were anesthetized, skin and muscle layers were cut open, the liver was exposed, a perfusion catheter was inserted into the portal vein, and the inferior vena cava was cut open to prepare for liver perfusion. Perfusion Solution I (Hank's, 0.5mM EGTA, pH 8) and perfusion Solution II (Low-glucose DMEM, 100U/mL Type IV, pH 7.4) were preheated at 40 deg.C and perfused into the liver along the portal vein cannula at 37 deg.C, flow rate 7mL/min, perfusion for 5min, until the liver became off-white. The liver was then perfused with a 37 ℃ perfusion Solution II at a flow rate of 7mL/min for 7 min. After perfusion was completed, the liver was removed and placed in Solution III (10% FBS low-glucose DMEM, 4 ℃) to stop digestion, the liver envelope was scratched with forceps, and the hepatocytes were released by gentle shaking. The hepatocytes were filtered through a 70 μm cell filter, centrifuged at 50g for 2min, and the supernatant was discarded. The cells were resuspended in Solution IV (40% percoll low-glucose DMEM, 4 ℃), centrifuged at 100g for 2min and the supernatant discarded. 2% FBS low-glucose DMEM was added to resuspend the cells for use. Trypan blue staining identifies cell viability.

2. Determination of GalNAc-siRNA binding curves and Kd values

Freshly isolated mouse primary hepatocytes were plated in 96-well plates at 2X 10 ⁴ One/well, 100. mu.L/well. Different GalNAc-siRNA was added to each well (see Table 2). Each GalNAc-siRNA was set at a final concentration of 0.9nM, 2.7nM, 8.3nM, 25nM, 50nM or 100 nM. After incubation at 4 ℃ for 2h, 50g was centrifuged for 2min and the supernatant was discarded. Cells were resuspended at 10. mu.g/mL PI, stained for 10min and centrifuged at 50g for 2 min. The cells were washed with pre-cooled PBS, centrifuged at 50g for 2min and the supernatant discarded. PBS resuspended cells. Flow cytometry (Beckman) determination of mean Fluorescence intensity MFI of viable cells (mean Fluorescence intensity), GraphPad Prism 5 software for non-linear fitting and dissociation constant K _d And (4) calculating the value.

The results are shown in Table 3. The data indicate that GalNAc-siRNA can specifically target hepatocytes; GalNAc ligands have Kd values between 1.5-27.6nM with cellular receptors and higher affinity compared to prior art galactose ligands, and RB-101, RB-104, RB-108 and RB-109 structures exhibit relatively stronger receptor affinity (the smaller the Kd value, the greater the affinity).

TABLE 3 Experimental groups K _d And K _d Value (nM)

Sample numbering	RB-101	RB-102	RB-103	RB-104	RB-105
						Kd	1.879	3.118	4.265	1.958	19.64
Ki	1.6	2.922	1.049	1.031	12.57
						Sample numbering	RB-106	RB-107	RB-108	RB-109	RB-110
Kd	4.632	2.066	1.874	1.982	6.371
						Ki	2.984	1.161	1.018	1.057	2.708

EXAMPLE 18 in vivo liver Targeted assay

The test adopts male SPF grade Balb/c-nu mice (purchased from Beijing Wintolite laboratory animals Co., Ltd.) with the age of 6-7 weeks, and the mice are randomly divided into 6 groups, namely a blank control group, NC1 (unconjugated ligand), a test group 1, a test group 2, a test group 3 and a test group 4. The number of animals in each group was 5, and the animals were administered by tail vein injection at a dose of about 10mg/kg (see Table 4 for experimental design). All animals were imaged in vivo before drug administration, 15min, 30min, 1h, 2h, 4h, 6h post drug administration, including white light imaging. After 6 hours euthanasia after drug administration, brain, salivary glands, heart, spleen, lung, liver, kidney and intestinal tract were removed for ex vivo organ imaging (brand: Bruker, model: XTREME).

TABLE 4 design of liver targeting experiments

Serial number	Group of	Test article	Administration dose (mg/kg)	Volume of administration (mL)
					1	Blank control group	Physiological saline	0	0.2
2	NC1	RB-100	10	0.2
					3	Test group 1	RB-104	10	0.2
4	Test group 2	RB-107	10	0.2
					5	Test group 3	RB-108	10	0.2
6	Test group 4	RB-110	10	0.2

Ex vivo imaging results are shown in table 5.

TABLE 5 statistical results of isolated organ fluorescence intensity values (mean total photon number p/sec) after background subtraction

The results show that: the RB-100, RB-104, RB-107, RB-108 and RB-110 test substances are mainly distributed in the liver, the kidney and the gastrointestinal tract, and are less distributed in tissues such as the brain, the heart, the lung, the spleen and the like.

When compared with RB-100 group (negative control group), the statistical result of the average total photon number shows that the RB-104, RB-107, RB-108 and RB-110 test substances have the liver targeting effect. Comparing the fluorescence intensity of the test objects in different organs, wherein the statistical difference of the liver fluorescence intensity of the RB-107 test objects and the RB-108 test objects is extremely obvious (P <0.001), the statistical difference of the liver fluorescence intensity of the RB-104 test objects is obvious (P <0.01), and the statistical difference of the liver fluorescence intensity of the RB-110 test objects is obvious (P < 0.05).

The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference in their entirety. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Accordingly, other embodiments are within the scope of the following claims.

Sequence listing

<110> Yangzhou Ribo Biotech Co., Ltd

Agena Biopharmaceutical Co., Ltd.

<120> Compounds, conjugates and uses thereof

<130> C20P8019

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> exemplary antisense strand sequences

<220>

<221> modified base

<222> (1)..(3)

<223> 2' OMe modification per base

<220>

<221> modified base

<222> (4)..(4)

<223> 2' F modification

<220>

<221> modified base

<222> (5)..(7)

<223> 2' OMe modification per base

<220>

<221> modified base

<222> (8)..(8)

<223> 2' F modification

<220>

<221> modified base

<222> (9)..(11)

<223> 2' OMe modification per base

<220>

<221> modified base

<222> (12)..(12)

<223> 2' F modification

<220>

<221> modified base

<222> (13)..(14)

<223> 2' OMe modification per base

<220>

<221> modified base

<222> (15)..(15)

<223> 2' F modification

<220>

<221> modified base

<222> (16)..(16)

<223> 2' OMe modification

<220>

<221> modified base

<222> (17)..(17)

<223> 2' F modification

<220>

<221> modified base

<222> (18)..(18)

<223> 2' OMe modification

<220>

<221> modified base

<222> (19)..(19)

<223> 2' F modification

<400> 1

ugacaaacgg gcaacauac 19

<210> 2

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> example sense Strand sequences

<220>

<221> modified base

<222> (1)..(1)

<223> 2' OMe modification

<220>

<221> modified base

<222> (2)..(2)

<223> 2' F modification

<220>

<221> modified base

<222> (3)..(3)

<223> 2' OMe modification

<220>

<221> modified base

<222> (4)..(4)

<223> 2' F modification

<220>

<221> modified base

<222> (5)..(5)

<223> 2' OMe modification

<220>

<221> modified base

<222> (6)..(7)

<223> 2' F modification per base

<220>

<221> modified base

<222> (8)..(8)

<223> 2' OMe modification

<220>

<221> modified base

<222> (9)..(11)

<223> 2' F modification per base

<220>

<221> modified base

<222> (12)..(12)

<223> 2' OMe modification

<220>

<221> modified base

<222> (13)..(15)

<223> 2' F modification per base

<220>

<221> modified base

<222> (16)..(16)

<223> 2' OMe modification

<220>

<221> modified base

<222> (17)..(18)

<223> 2' F modification per base

<220>

<221> modified base

<222> (19)..(19)

<223> 2' OMe modification

<400> 2

guauguugcc cguuuguca 19

Claims

1. A compound of formula (I), or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof,

wherein R is ₁ Is a moiety that binds to asialoglycoprotein receptor (ASGPR);

R ₂ is-CH ₂ -、

C _6-14 Aryl radical, C _2-6 Alkenyl or C _2-6 An alkynyl group;

R ₃ is composed of

-O-、C _6-14 An aromatic group or a 3-to 10-membered heterocyclic group;

R ₄ is-CH ₂ -、

C _6-14 Aryl or 3 to 10 membered heterocyclyl;

R ₅ is-OH,

-COOH、

PG is a carboxyl protecting group;

PG ₂ is a hydroxy protecting group;

a. b, c and d are each independently any integer from 1 to 10;

n is any integer of 1-3.

2. A compound of claim 1, wherein R is ₁ The end of the part is galactose or N-acetylgalactosamine;

optionally, said R ₁ Moieties are selected from the following structures:

wherein q and q' are each independently any integer from 1 to 10.

3. A compound according to claim 1 or 2, wherein the carboxyl protecting group is selected from the group consisting of succinate, pentafluorophenol ester, and,

Wherein h is any integer from 1 to 5.

4. A compound according to any one of claims 1 to 3, wherein the hydroxyl protecting group is selected from silyl, monomethoxytrityl (MMTr), 4' -dimethoxytrityl (DMTr) and trityl; optionally, the silyl group is selected from tert-butyldimethylsilyl ether (TBMDS), tert-butyldiphenylsilyl (TBDPS), and triisopropylsilyl ether (TIPS).

5. The compound of claim 1, wherein the compound has the structure shown below:

6. a compound of formula (II), or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof,

wherein R is ₁ Is a moiety that binds to asialoglycoprotein receptor (ASGPR);

R ₂ is-CH ₂ -、

C _6-14 Aryl radical, C _2-6 Alkenyl or C _2-6 An alkynyl group;

a and b are each independently any integer from 1 to 10;

n is any integer of 1-3.

7. A compound of claim 6, wherein R is ₁ The end of the part is galactose or N-acetylgalactosamine;

optionally, said R ₁ Moieties are selected from the following structures:

wherein q and q' are each independently any integer from 1 to 10.

8. The compound of claim 6, wherein the compound has the structure shown below:

9. a conjugate having a structure represented by formula (III),

wherein,

R ₁ is a moiety that binds to ASGPR;

R ₂ is-CH ₂ -、

C _6-14 Aryl radical, C _2-6 Alkenyl or C _2-6 An alkynyl group;

R ₃ is composed of

-O-、C _6-14 An aromatic group or a 3-to 10-membered heterocyclic group;

R ₄ is-CH ₂ -、

C _6-14 Aryl or 3 to 10 membered heterocyclyl; r is O or S;

rx is a drug molecule selected from the group consisting of cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, toxins, radioisotopes, and oligonucleotide chains;

a. b, c and d are each independently any integer from 1 to 10;

n is any integer of 1-3.

10. The conjugate of claim 9, having a structure of formula (IV),

wherein the oligo is an oligonucleotide chain,

optionally, the oligonucleotide strand is a single-stranded oligonucleotide or a double-stranded oligonucleotide or a combination thereof.

11. A conjugate formed by linking a compound of any one of claims 1-8 to a drug molecule selected from the group consisting of cytotoxic agents, chemotherapeutic agents, growth inhibitory agents, toxins, radioisotopes, and oligonucleotide chains.

12. The conjugate of claim 11, wherein the drug molecule is an oligonucleotide chain,

13. The conjugate of any of claims 9-12, wherein the oligonucleotide strand comprises unmodified nucleotides and/or modified nucleotides.

14. The conjugate of claim 13, wherein the modified nucleotides are each independently selected from the group consisting of 2' -methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-allyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxy modified nucleotides, 2' -hydroxy modified nucleotides, locked nucleotide modified nucleotides, Glycerol Nucleic Acid (GNA) modified nucleotides, and Unlocked Nucleic Acid (UNA) modified nucleotides.

15. The conjugate of claim 16, wherein the 2 '-O-alkyl modification is a 2' -O-methyl modification.

16. The conjugate of any of claims 9-15, wherein the oligonucleotide chain has a terminal modification, optionally wherein the terminal modification is selected from cholesterol, polyethylene glycol, a fluorescent probe, biotin, a polypeptide, a vitamin, a tissue targeting molecule, or a combination thereof.

17. The conjugate of any one of claims 9-16, wherein the oligonucleotide strand is 5-100bp in length.

18. The conjugate of any of claims 9-17, wherein the compound is linked to the 5 'terminus, the 3' terminus, or any nucleotide between the 5 'terminus and the 3' terminus of at least one strand of the oligonucleotide strand by a phosphoester linkage.

19. The conjugate of claim 18, wherein the phosphate ester linkage is a phosphodiester linkage or a modified phosphate linkage.

20. The conjugate of claim 19, wherein the modified phosphate ester linkage is selected from the group consisting of a thio-modified phosphate ester linkage and an amino-modified phosphate ester linkage.

21. The conjugate according to any of claims 9-17, wherein the ribose-phosphate backbone of the oligonucleotide strand is substituted with a Polypeptide Nucleic Acid (PNA), or a morpholine ring antisense nucleotide (PMO).

22. The conjugate according to any one of claims 9 to 21, wherein the conjugate is obtained by solid phase synthesis or solution phase synthesis.

23. A method for preparing a compound according to any one of claims 6 to 8, comprising reacting pentaerythritol and NaN ₃ And galactose or a galactose derivative is subjected to a synthesis reaction to obtain the compound.

24. A process for the preparation of a compound according to any one of claims 1 to 5, wherein a first compound is contacted with a second compound to obtain said compound, wherein said first compound is as defined in any one of claims 6 to 8 or obtained according to the process of claim 23, and wherein said second compound has the structure shown below:

wherein c and d are each independently any integer from 1 to 10.

25. A composition, comprising: the conjugate of any one of claims 9-22, optionally further comprising a pharmaceutically acceptable carrier or excipient.

26. The composition of claim 25, wherein the composition is in the form of a powder, tablet, granule, capsule, solution, emulsion, suspension, injection, spray, aerosol, powder spray, or microneedle patch.

27. The composition of claim 25, wherein the composition is administered to the subject intravenously, intramuscularly, or subcutaneously;

optionally, the subject is a mammal;

optionally, the mammal is selected from the group consisting of bovines, equines, ovines, porcines, canines, felines, rodents, and primates.

28. Use of a conjugate according to any of claims 9-22 in the manufacture of a medicament for the treatment and/or prevention of a pathological condition or disease associated with expression or overexpression of a gene in a hepatocyte.

29. The use according to claim 28, wherein said gene is selected from the group consisting of HBV genome, HCV genome, PCSK9, xanthine oxidase, URAT1, APOB, liver fibrosis related genes (AP3S2, AQP2, AZINl, DEGSl, STXBP5L, TLR4, TRPM5, etc.), non alcoholic steatohepatitis (PNPLA3, FDFTl), primary biliary cirrhosis (HLA-DQB1, IL-12RB2, etc.), or a combination thereof.

30. The use according to claim 28, wherein the disease is selected from hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, alpha-1-antitrypsin deficiency, bile acid synthesis and metabolic defects, biliary atresia, cystic fibrosis liver disease, idiopathic neonatal hepatitis, mitochondrial liver disease, progressive familial intrahepatic cholestasis, primary sclerosing cholangitis, transthyretin amyloidosis, hemophilia, homozygous familial hypercholesterolemia, hyperlipidemia, Hepatitis B (HBV), Hepatitis C (HCV), steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia or a disease involving abnormally increased hepatic glucogenesis similar to type II diabetes, hepatitis and hepatic porphyrins.

31. Use of a compound according to any one of claims 1 to 8 or a conjugate according to any one of claims 9 to 22 in the preparation of a composition for liver targeting.

32. Use of a conjugate according to any of claims 9 to 22 in the preparation of a composition for liver-targeted RNA detection or localization.

33. A method for intervening or detecting a predetermined gene in a hepatocyte, wherein a population of cells comprising the hepatocyte is contacted with the conjugate of any one of claims 9 to 22, wherein the oligonucleotide strand in the conjugate interacts with the predetermined gene.